A major problem in medicine has been the development of drug resistant bacteria as more antibiotics are used for a wide variety of illnesses and other conditions. Hospital infections are the 8th leading cause of death in the United States, due in large part to drug-resistant and newly-emerging pathogens. The use of more antibiotics and the number of bacteria showing resistance has prompted longer treatment times. Furthermore, broad, non-specific antibiotics, some of which have detrimental effects on the patient, are now being used more frequently. A related problem with this increased use is that many antibiotics do not penetrate mucus linings easily. Additionally, the number of people allergic to antibiotics appears to be increasing. Accordingly, there is a commercial need for new antibacterial approaches, especially those that operate via new modalities or provide new means to kill pathogenic bacteria.
Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharide. The gram-positive cell wall appears as a broad, dense wall that is 20-80 nm thick and consists of numerous interconnecting layers of peptidoglycan. Between 60% and 90% of the gram-positive cell wall is peptidoglycan, providing cell shape, a rigid structure, and resistance to osmotic shock. The cell wall does not exclude the Gram stain crystal violet, allowing cells to be stained purple, and therefore “Gram-positive.” Gram-positive bacteria include but are not limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, and Clostridium. Medically relevant species include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. 
Antibacterials that inhibit cell wall synthesis, such as penicillins and cephalosporins, interfere with the linking of the interpeptides of peptidoglycan and weaken the cell wall of both gram positive and gram negative bacteria. Because the peptidoglycans of gram-positive bacteria are exposed, gram-positive bacteria are more susceptible to these antibiotics. Advantageously, eukaryotic cells lack cell walls and are not susceptible to these drugs or other cell wall agents.
Attempts have been made to treat bacterial diseases through the use of bacteriophages. However, the direct introduction of bacteriophages into an animal to prevent or fight diseases has certain drawbacks. Specifically, both the bacteria and the phage have to be in the correct and synchronized growth cycles for the phage to attach. Additionally, there must be the right number of phages to attach to the bacteria; if there are too many or too few phages, there will be either no attachment or no production of the lysing enzyme. The phage must also be active enough. The phages are also inhibited by many things including bacterial debris from the organism it is going to attack. Further complicating the direct use of a bacteriophage to treat bacterial infections is the possibility of immunological reactions, rendering the phage non-functional.
Novel antimicrobial therapy approaches include enzyme-based antibiotics (“enzybiotics”) such as bacteriophage lysins. Phages use these lysins to digest the cell wall of their bacterial hosts, releasing viral progeny through hypotonic lysis. A similar outcome results when purified, recombinant lysins are added externally to Gram-positive bacteria. The high lethal activity of lysins against Gram-positive pathogens makes them attractive candidates for development as therapeutics. Bacteriophage lysins were initially proposed for eradicating the nasopharyngeal carriage of pathogenic streptococci (Loeffler, I. M. et al (2001) Science 294: 2170-2172; Nelson, D. et al (2001) Proc Natl Acad Sci USA 98:4107-4112). Lysins are part of the lytic mechanism used by double stranded DNA (dsDNA) phage to coordinate host lysis with completion of viral assembly (Wang, I. N. et al (2000) Annu Rev Microbiol 54:799-825). Phage encode both holins that open a pore in the bacterial membrane, and peptidoglycan hydrolases called lysins that break bonds in the bacterial wall. Late in infection, lysin translocates into the cell wall matrix where it rapidly hydrolyzes covalent bonds essential for peptidoglycan integrity, causing bacterial lysis and concomitant progeny phage release.
Lysin family members exhibit a modular design in which a catalytic domain is fused to a specificity or binding domain (Lopez, R. et al (1997) Microb Drug Resist 3:199-21 1). Lysins can be cloned from viral prophage sequences within bacterial genomes and used for treatment (Beres, S. B. et al (2007) PLoS ONE 2(8): 1-14). When added externally, lysins are able to access the bonds of a Gram-positive cell wall (FIG. 1) (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400). Lysins have been shown to demonstrate a high lethal activity against numerous Gram-positive pathogens (especially the bacterium from which they were cloned), raising the possibility of their development as therapeutics (Fischetti, V. A. (2008) Curr Opinion Microbiol 11:393-400; Nelson, D. L. et al (2001) Proc Natl Acad Sci USA 98:4107-4112).
Bacteriophage lytic enzymes have been established as useful in the assessment and specific treatment of various types of infection in subjects through various routes of administration. For example, U.S. Pat. No. 5,604,109 (Fischetti et al.) relates to the rapid detection of Group A streptococci in clinical specimens, through the enzymatic digestion by a semi-purified Group C streptococcal phage associated lysin enzyme. This enzyme work became the basis of additional research, leading to methods of treating diseases. Fischetti and Loomis patents (U.S. Pat. Nos. 5,985,271, 6,017,528 and 6,056,955) disclose the use of a lysin enzyme produced by group C streptococcal bacteria infected with a CI bacteriophage. U.S. Pat. No. 6,248,324 (Fischetti and Loomis) discloses a composition for dermatological infections by the use of a lytic enzyme in a carrier suitable for topical application to dermal tissues. U.S. Pat. No. 6,254,866 (Fischetti and Loomis) discloses a method for treatment of bacterial infections of the digestive tract which comprises administering a lytic enzyme specific for the infecting bacteria. The carrier for delivering at least one lytic enzyme to the digestive tract is selected from the group consisting of suppository enemas, syrups, or enteric coated pills. U.S. Pat. No. 6,264,945 (Fischetti and Loomis) discloses a method and composition for the treatment of bacterial infections by the parenteral introduction (intramuscularly, subcutaneously, or intravenously) of at least one lytic enzyme produced by a bacteria infected with a bacteriophage specific for that bacteria and an appropriate carrier for delivering the lytic enzyme into a patient.
Phage associated lytic enzymes have been identified and cloned from various bacteriophages, each shown to be effective in killing specific bacterial strains. U.S. Pat. Nos. 7,402,309, 7,638,600 and published PCT Application WO2008/018854 provides distinct phage-associated lytic enzymes useful as antibacterial agents for treatment or reduction of Bacillus anthracia infections. U.S. Pat. No. 7,569,223 describes the pneumococcal phage lytic enzyme Pal for Streptococcus pneumoniae. Lysin useful for Enterococcus (E. faecalis and E. faecium, including vancomycin resistant strains) are described in U.S. Pat. No. 7,582,291. US 2008/0221035 describes mutant Ply GBS lysins highly effective in killing Group B streptococci. A chimeric lysin denoted ClyS, with activity against Staphylococci bacteria, including Staphylococcus aureus, is detailed in WO 2010/002959.
Streptococcus pneumoniae (S. pneumoniae), a gram-positive encapsulated diplococcus, is a primary etiologic agent in human illnesses such as bacteremia, meningitis, pneumonia, otitis media, and sinusitis. This bacterium is responsible for the death of >1 million children per year under five years of age worldwide (English, M (2000) Paediatr Respir Rev 1:21-5) and community-acquired pneumonia is the sixth most common cause of death in the USA (File, T M (2004) Am J Med 117 Suppl 3A:39S-50S). Moreover, S. pneumoniae is a major cause of acute otitis media worldwide, a disease that affects more than 5 million children per year in the USA (CDC (2009) Pneumococcal diseases, p. 217-30. In W. Atkinson, et al (ed.), Epidemiology and prevention of vaccine-preventable diseases (11th ed), Public Health Foundation, Washington DC). Finally, secondary infections as a result of influenza pandemics account for >90% of deaths, with S. pneumoniae being the leading cause of these deaths (Brundage, J F Shanks G D (2008) Emerg Infect Dis 14:1193-9; Brundage, J F Shanks G D (2007) J Infect Dis 196:1717-8; Morens, D M et al (2009) N Engl J Med 361:225-9; Morens, D M et al (2009) Public Health Rep 124:22-5). Pneumococcal infections are often treated with antibiotics, but bacteriologically confirmed treatment failures, due to the increasing incidence of resistance (Reinert, R R (2009) Clin Microbiol Infect 15 Suppl 3:1-3) were reported for macrolides, fluoroquinolones, and cephalosporins (Mandell, L A et al (2002) Clin Infect Dis 35:721-7). The overuse and misuse of antibiotics as a result of treatment of millions of otitis media cases only contribute to the emergence of resistant strains (Goossens, H (2009) Clin Microbiol Infect 15 Suppl 3:12-5. Taken together, these observations have prompted the need for new drugs, acting by totally different mechanisms, for the treatment and prevention of pneumococcal associated diseases.
Prior to the discovery of antibiotics, phages, a major predator of bacteria in nature, were viewed as a possible method to control pathogenic bacteria. At the time, several reports of the successful use of phage to treat infections were published (Sulakvelidze, A and Barrow, P (2005) Phage therapy in animals and agribusiness, p. 335-71. In E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: Biology and Applications, CRC Press, USA; Sulakvelidze, A and Kutter, E (2005) Bacteriophage therapy in humans, p. 381-426. In E. Kutter and A. Sulakvelidze (ed.), Bacteriophages: Biology and Applications, CRC Press, USA), but the advent of antibiotics in the 40's led to a rapid decline of phage therapy research in the western world. The past decade has seen a renewed interest in phage therapy and phage derived anti-bacterial compounds (Borysowski, J et al (2006) Exp Biol Med 231:366-77; Fischetti, V A (2008) Curr Opin Microbiol 11:393-400).
One of these products, phage endolysins or lysins, have been exploited for their rapid killing action on gram-positive bacteria (Borysowski, J et al (2006) Exp Biol Med 231:366-77; Fischetti, V A (2008) Curr Opin Microbiol 11:393-400). These specific enzymes are produced at the time when phage progeny need to escape the bacterial host. Pneumococcal phage Cp-1 produces the lysin Cpl-1, a 37 kDa enzyme. This lysin is constructed like all such endolysins, having two well defined domains connected by a flexible linker. The catalytic activity is restricted to the N-terminal domain, while the C-terminal part, containing 6 choline-binding repeats (ChBR) and a C-terminal tail of 13 amino-acids, is required for substrate binding in the pneumococcal cell wall. Cpl-1 belongs to the family of lysozymes which target the β1,4 linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan (Perez-Dorado, INE et al (2007) J Biol Chem 282:24990-9). Its choline-dependent activity makes Cpl-1 highly specific for S. pneumonia (Garcia, J L et al (1987) J Virol 61:2573-80). The Cpl-1 gene has been cloned into the high expression vector pin111An and then over-expressed and purified (Loeffler, J M et al (2003) Infect Immun 71:6199-204).
Purified Cpl-1 has been successfully tested for treating pneumococcal sepsis (Jado, I et al (2003) J Antimicrob Chemother 52:967-73; Loeffler, J M and Fischetti, V A (2003) Antimicrob Agents Chemother 47:375-7), endocarditis (Entenza, J M et al (2005) Antimicrob Agents Chemother 49:4789-92), pneumococcal meningitis (Grandgirard, D et al (2008) J Infect Dis 197:1519-22), and pneumonia (Witzenrath, M et al (2009) Crit Care Med 37:642-9) in rodent models. Nevertheless, proteins are usually quickly cleared in vivo and repeated injections or even continuous infusion of Cpl-1 was required in many of the studies performed to date (Entenza, J M et al (2005) Antimicrob Agents Chemother 49:4789-92. 31; Witzenrath, M et al (2009) Crit Care Med 37:642-9).
These results may be a shortcoming for the clinical development of Cpl-1 and similar lysins. What is needed in the art are improved lysins that may be used to treat pneumococcal diseases having killing activity and enhanced clinically-relevant parameters, such as longer half life or reduced clearance in vivo.